ABSTRACT
Non-segmented negative-strand RNA viruses, including Ebola virus (EBOV), rabies virus, human respiratory syncytial virus and pneumoviruses, can cause respiratory infections, haemorrhagic fever and encephalitis in humans and animals, and are considered a substantial health and economic burden worldwide1. Replication and transcription of the viral genome are executed by the large (L) polymerase, which is a promising target for the development of antiviral drugs. Here, using the L polymerase of EBOV as a representative, we show that de novo replication of L polymerase is controlled by the specific 3' leader sequence of the EBOV genome in an enzymatic assay, and that formation of at least three base pairs can effectively drive the elongation process of RNA synthesis independent of the specific RNA sequence. We present the high-resolution structures of the EBOV L-VP35-RNA complex and show that the 3' leader RNA binds in the template entry channel with a distinctive stable bend conformation. Using mutagenesis assays, we confirm that the bend conformation of the RNA is required for the de novo replication activity and reveal the key residues of the L protein that stabilize the RNA conformation. These findings provide a new mechanistic understanding of RNA synthesis for polymerases of non-segmented negative-strand RNA viruses, and reveal important targets for the development of antiviral drugs.
Subject(s)
Ebolavirus , RNA, Viral , RNA-Dependent RNA Polymerase , Virus Replication , Animals , Humans , Antiviral Agents/pharmacology , Ebolavirus/enzymology , Ebolavirus/genetics , Ebolavirus/growth & development , Hemorrhagic Fever, Ebola/virology , RNA, Viral/biosynthesis , RNA, Viral/chemistry , RNA, Viral/genetics , RNA, Viral/metabolism , RNA-Dependent RNA Polymerase/antagonists & inhibitors , RNA-Dependent RNA Polymerase/metabolism , Genome, Viral , Nucleic Acid Conformation , Mutagenesis , RNA StabilityABSTRACT
Proparacaine is a widely used topical anesthetic in ophthalmic optometry and surgery, and has been reported to have cytotoxic effects on rabbit corneal endothelial cells after prolonged and repeated usage. Since rabbit is an exceptive mammal whose corneal endothelial cells still maintaining proliferation abilities even in adulthood, whether proparacaine has cytotoxic effects on human corneal endothelial (HCE) cells need to be further verified. Our objectives in the present study were to investigate the cytotoxicity to HCE cells of proparacaine and its underlying mechanisms in vitro and verify the cytotoxicity using cat corneal endothelial (CCE) cells in an in vivo model of cat corneas. Cytotoxic evaluation results indicated that a dose- and time-dependent toxic response of HCE cells to proparacaine over 0.03125% was rated based on morphology and viability, and a toxic response of CCE cells to 0.5% (clinical applied dosage) proparacaine was also rated based on cell density and histology. Importantly, treatment with proparacaine resulted in significant elevation of plasma membrane permeability, cell cycle arrest at S phase, fragmentation of genomic DNA, formation of apoptotic bodies, and externalization of phosphatidylserine (PS) of HCE cells. Moreover, proparacaine demonstrated disrupting effects on mitochondrial transmembrane potential (MTP) of HCE cells and activating effects on caspase-3, -8 and -9. This study demonstrates that proparacaine has notable cytotoxicity to both HCE cells in vitro and CCE cells in vivo, and its dose- and time-dependent cytotoxicity to HCE cells is achieved by inducing apoptosis via a mitochondrion-mediated caspase-dependent pathway. These findings provide new insights into the cytotoxicity and apoptosis-inducing effect of local anesthetics which should be used with great caution in the eye clinic.
